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KMID : 1094720110160020297
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Biotechnology and Bioprocess Engineering
2011 Volume.16 No. 2 p.297 ~ p.297
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Improved production and properties of β-glucosidase influenced by 2-deoxy-d-glucose in the culture medium of Termitomyces clypeatus
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Ghorai Shakuntala
Mukherjee Sumana
Khowala Suman
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Abstract
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Increased production, secretion, and activity of β-glucosidase in the filamentous fungus Termitomyces clypeatus was achieved in presence of the glycosylation inhibitor 2-deoxy-d-glucose (0.05%, w/v) during submerged fermentation. Enzyme activity increased to 163 U/mL by adding mannose (2 mg/mL) to the medium. Such a high enzyme activity has not been achieved without mutation or genetic manipulation. The Km and Vmax of the enzyme in culture medium were determined to be 0.092 mM and 35.54 U/mg, respectively, with p-nitrophenyl β-d-glucopyranoside as substrate, confirming its high catalytic activity. The enzyme displayed optimum activity at pH 5.4 and 45°C. The enzyme was fairly stable between acidic to alkaline pH and retained about 75 ∼ 65% residual activities between pH 4 and 10.6 and demonstrated full activity at 45°C for 3 days. The enzyme was also stable in the presence of Zn2+ and Mg2+ and 80% of the residual activity was observed in the presence of Mn2+, Ca2+, K+, Cu2+, EDTA, and sodium azide. Around 70% of the activity was retained in the presence of 2 M guanidium HCl and 3 M urea, whereas the activity was 5 and 2 times higher in the presence of 4 mM beta-mercaptoethanol and 50 mM DTT, respectively. The enzyme obtained from the culture filtrate showed potential cellulose saccharifying ability which increased further when supplemented with commercial cellulase. Thus, this enzyme could be used without any additional downstream processing for commercial cellulase preparation and production of bioethanol or for other biotechnological applications.
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KEYWORD
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β-glucosidase, cellulolytic enzymes, 2-deoxy-d-glucose, Termitomyces clypeatus, saccharification
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